Production of stable high-potency human ahf using polyethylene glycol and glycine to fractionate a cryoprecipitate of ahf concentrate

ABSTRACT

THE PRODUCTION OF A STABLE ANTIHEMOPHILIC FACTOR OF HIGH POTENCY BY THE FRACTIONATION OF A CRYOPRECIPITATE CONCENTRATE OF ANTIHEMOPHILIC FACTOR.

United States Patent O ABSTRACT OF THE DISCLOSURE The production of astable antihemophilic factor of high potency by the fractionation of acryoprecipitate concentrate of antihemophilic factor.

This application is a continuation of application Ser. No. 679,240,filed Oct. 30, 1967, now abandoned, which was a continuation-in-part ofapplication Ser. No. 634,839, filed May 1, 1967, and now abandoned.

This invention relates to a method of making a concentrate ofantihemophilic factor (AHF, Factor VIII).

The process of blood coagulation is an important activity which normalwhole blood is capable of carrying out under timely circumstances toprevent excess loss of blood through open wounds or by internalbleeding. It is known that normal whole blood contains a factor which isabsent or seriously deficient in hemophiliacs. This factor is associatedwith the globulin fraction of blood and has come to be known asantihemophilic factor (AHF, Factor VIII).

Scientists have known about AHF and its role in blood coagulation forsome time, and treatment of hemophilia heretofore has generallyconsisted of replacement therapy whereby the patient is transfused withmany pints of fresh whole blood or specially prepared plasma.

It is known, however, that under ordinary storage conditions whole bloodand liquid plasma lose their AHF activity in a day or so. While it ispossible to freeze and store fresh plasma, AHF activity in frozen plasmahas a relatively short life of several months or so.

It is also possible to decrease the loss of AHF activity during storageby drying freshly frozen plasma, but it has not heretofore been possibleto know with certainty the strength of AHF in this dried material sincethe amount of AHF in normal individuals who donate the plasma varieswidely, from about 50% to about 200% of average.

Conventional replacement therapy also suffers from other seriousdisadvantages since the hemophiliac often develops allergic orrefractory states when repeatedly treated with plasma. Moreover, thelarge amounts of plasma needed by the hemophiliac tend to causehypervolemia or overloading of the circulatory system. Such overloadingputs a strain on the heart and threatens the patient with heart failure.

Scientists have also proposed various methods for the isolation of AHFor the preparation of plasma fractions rich in AHF from human or animalblood. In practice, these methods heretofore have provided to beunreliable since the AHF activity of the fractions tends to be lostduring the isolation. AHF is a labile trace protein which is difiicultto separate completely from other plasma proteins, particularlyfibrinogen, and the yield of AHF from the plasma by these previousmethods has not been high. The AHF potencies of previously preparedblood 3,631,.18 Patented Dec. 28, 1971 fractions for hemophiliac therapyare known to be low and the cost of treatment is high.

Other drawbacks in prior attempts to isolate AHF are that AHF is readilysusceptible to denaturation by heat, freezing and thawing, and continuedstorage.

Among the various methods that have been heretofore proposed for theisolation of AHF are chromatography, batch absorption and elution, andselective precipitation. Various precipitating agents that have beenused are ethanol, ethyl ether, ammonium sulfate, phosphate-sodiumcitrate, amino acids, and cryoprecipitation procedures. Recently, theclinical use of a glycine-precipitated AHF fraction of whole plasma wasdisclosed by Webster et al., Amer. J. Med. Sciences, vol. 250, No. 6,pp. 643-650 (1965), and the clinical trials of AHF replacement therapywith a cryoprecipitated fraction of whole plasma in a closed-bag systemwas reported by Pool et al., New England J. Med., vol. 273, No. 27, pp.1443l447 (1965). None of the above methods, however, has proved to be acompletely practical method for isolating AHF.

In accordance with the present invention, as distinguished from theprior art, a method for the preparation of a stable AHF of high potencyis provided which comprises the fractionation of a cryoprecipitateconcentrate of human or animal AHF.

As used herein the term cryoprecipitate concentrate of AHF refers to theprecipitate obtained from the freezing of human or animal blood plasmaat 4 C. and separated from the supernatant. It is preferred that thiscryoprecipitate concentrate of AHF is obtained by rapid freezing offresh plasma, but stored plasma can also be used as source material inthe practice of the present invention. It is also preferred to carry outthe freezing to a temperature of from about 20 C. to about 40" C.followed by slow thawing to about 4 C.

In addition to the stability and high potency of AHF activity obtainedby the method of the present invention, use of the cryoprecipitateconcentrate of AHF rather than Whole plasma as the starting material hasthe advantage of allowing retention of other plasma components such asalbumin, gamma globulin and the like which are usually destroyed whenwhole plasma is purified by previously known methods of preparing AHFfractions for use in treatment of hemophilia.

In accordance with the present invention the cryoprecipitate concentrateof AHF preferably is fractionated to yield a stable AHF of high potencyby means comprising one or more of the following steps:

(a) precipitation with glycine, (b) precipitation with polyethyleneglycol.

In the glycine precipitation step, the cryoprecipitate concentrate ofAHF is first redissolved and then the redissolved fraction isprecipitated with an aqueous solution of glycine having a molarity offrom about 1.3 to about 1.8, followed by recovery and redissolution ofthe precipitate.

Recovery of the cryoprecipitate concentrate and the glycine-precipitatedfraction for use in this invention can be accomplished, for example, bycentrifugation or filtration of the respective precipitates or bysimilar such procedures.

Redissolution of the recovered precipitates can be achieved by warmingand agitating in citrated saline solution. In the case of theredissolution of the cryoprecipitate concentrate, it is preferred to usea glycine citrated saline solution and to increase the volume of thecryoprecipitate concentrate to about one-tenth the volume of originalplasma the cryoprecipitate concentrate represents. Theglycine-precipitated fraction is preferably redissolved with citratedsaline solution to increase the volume of the frac- 3 tion to aboutone-twentieth the volume of plasma the glycine-precipitated fractionrepresents.

It is also preferred to purify each of the respective redissolvedcryoprecipitate concentrate and glycine-precipitated fraction byclarifying with additional centrifugation and/or filtration to removeany insoluble matter.

The above fractionation of a cryoprecipitate concentrate of AHF byprecipitation with glycine has been found to provide an AHF concentrateof high potency which can be frozen and rendered stable, such as bylyophilization, and retained under ordinary refrigeration conditions forperiods of a year or longer. The potency of each batch of materialprepared by the above fractionation method can be precisely determinedso that the administering physician can know for the first time exactlyhow much AHF his patient receives.

Since the redissolved AHF concentrate prepared by the abovefractionation method has more than five times the AHF activity of anequal volume of plasma, the hemophiliac can be given a quantity of AHFwhich the heart could not otherwise tolerate. Even more importantly, theAHF activity in the above-prepared concentrate is contained in less thanone-fifteenth the amount of protein present in plasma providing an equalamount of AHF activity. This lower protein content minimizes thelikelihood of allergic reactions by the hemophiliac recipient andreduces the possibility of overloading the circulatory system.

Another example of means which can be used to fractionate thecryoprecipitate concentrate of AHF according to the present invention isprecipitation with polyethylene glycol. Polyethylene glycols are highmolecular weight polymers which are generally produced by reactingethylene oxide with ethylene glycol or water and have the followingstructure:

in which n represents the average number of oxyethylene groups.According to the present invention the polyethylene glycols should benontoxic and can range in molecular weight from about 200 to about20,000. They preferably have molecular weights from about 400 to about6,000. PEG 4000, which is a polyethylene glycol product having anaverage molecular weight of about 4,000, is the preferred product ofthis group.

In the polyethylene glycol precipitation step it is preferred to usefrom about 3% to about 4% polyethylene glycol by weight of theredissolved AHF concentrate with retention of the supernate, followed byuse of about polyethylene glycol by weight of the supernate with restep.If the polyethylene glycol precipitation step precedes the glycineprecipitation step, the molarity of the glycine used for theprecipitation should be about 1.8.

In the aforesaid combination of precipitation steps, it is preferredthat the glycine precipitation step follow the polyethylene glycolprecipitation step. It has been found that the glycine helps to removeany residual polyethylene glycol remaining in the concentrate of AHF andthereby reduces any potential danger from the presence of polyethylneglycol. Moreover, the concentrate of A'HF prepared in this manner hasbeen found to be substantially more soluble than the like product inwhich the glycine precipitation step preceded the polyethylene glycolprecipitation step.

The AHF concentrate prepared by the successive precipitation steps withglycine and polyethylene glycol as hereinbefore described can be frozenand rendered stable, such as by lyophilization, and retained underordinary refrigeration conditions for periods of a year or longer. Thereconstituted AHF concentrate has more than thirty times the AHFactivity of an equal volume of plasma; and

the AHF activity of this concentrate is contained in less than onehundredth the amount of protein present in plasma providing an equalamount of AHF activity.

The cryoprecipitate concentrate of AHF which has been fractionated byglycine precipitation and/or polyethylene glycol precipitation can befurther treated by purification with ECTEOLA cellulose resin. Thispurification can be carried out either before or after the glycine and/or polyethylene glycol precipitation and may be done by column or batchtechniques. The concentrate purified by this method has the additionaladvantage that it can also be administered intramuscularly as well as bythe intravenous administration methods generally used in the case of theAHF concentrate which has not been treated with the ECTEOLA celluloseresin. The AHF concentrate purified with ECTEOLA cellulose resin hasbeen found to be free of fibrinogen by the addition of thrombin and byimmunoelectrophoresis.

As used herein, the term ECTEOLA cellulose resin refers to a modifiedcellulose which contains active basic substituents introduced into thecellulose molecule by reaction with epichlorohydrin and triethanolamine.Methods of preparation of ECTEOLA cellulose resins are described ingeneral by Sober and Peterson, J. Am. Chem. Soc., vol. 76, pp. 1711-12(1956); id., vol. 78, pp. 751-55 (1956); vol. 78, pp. 756-63 (1956); andPeterson and Sober, Biochem. Preparations, vol. 8, pp. 43-4 (1961).

ECTEOLA cellulose resins are available commercially. However, it hasbeen found desirable to initially treat these resins by recycling themwith caustic soda before use in the herein-defined purificationprocedure.

In the purification procedure with ECTEOLA cellulose resin, the resinpreferably is first equiliberated with a chloride buffer solution havinga concentration of about 0.8% NaCl and then poured into achromatographic glass column. The AHF concentrate which is desired to bepurified is then applied to the column and finally eluted with achloride buffer solution having a molarity of about 0.5.

Other methods of purifying the AHF concentrate of the present inventionwill be apparent to those skilled in the art.

The following examples further illustrate the present invention althoughthe invention is not limited to these specific examples which areprovided for purposes of illustration and not limitation. All parts andpercentages herein are on a weight basis unless otherwise specified.

EXAMPLE 1 A stable human AHF concentrate of high potency is prepared bythe successive fractionation of human blood plasma, first bycryoprecipitation and then by glycine precipitation in the followingmanner:

Reagents Citrated saline.-One part 0.1 molar sodium citrate to fourparts by weight 0.9 percent saline.

Glycine citrated saline.Sufficient glycine is added to the abovecitrated saline to make a 0.1 molar solution respective of glycine.

Buffered wash water.To distilled water add volume of buffered citratewhich is made by adjusting 0.5 molar sodium citrate with 0.5 molarcritic acid to pH 6.88.

Acetic acid-Prepared both 1.0 normal and 0.1 normal aqueous solutions.

Glycine-Prepare 1.3 molar aqueous solution.

Procedure Human blood plasma is received frozen (54 C.) from a donorcenter. The plasma is pooled into Pfaudler kettles and, while held at atemperature of less than 4 C., it is centrifuged by continuous flow orbucket centrifugation. The resulting cryoprecipitate is collected andretained for further fractionation hereinafter in accordance with thepresent invention.

To the cryoprecipitate, glycine citrated saline is added, the amountbeing one-tenth the volume of plasma the cryoprecipitate represents.Dissolution is brought about by mixing the cryoprecipitate and glycinecitrated saline in a warm environment (room temperature, but not inexcess of 30 C.).

When the cryoprecipitate has dissolved, it can be clarified, if desired(depending upon the amount of red cells and denatured protein present),by further centrifugation and/ or filtration.

The dissolved cryoprecipitate is then adjusted to pH 6.88 with 0.1normal acetic acid. By suitable means (for example, refrigeration or useof isopropanol Dry (Ice bath) the solution is cooled to a temperature offrom 6 C. to C. To the cooled solution, suflicient glycine is added tomake the solution 1.2 molal with respect to glycine. The mixture isgently agitated for 45 to 60 minutes at a temperature of from 2 C. to 10C., and then centrifuged by continuous flow or bucket centrifugation.The resulting glycine precipitate is collected and gently washed withbuffered water at a temperature of 0 to 4 C.

When the glycine precipitate has dissolved, it is preferable to clarifythe solution by centrifugation and/or filtration using a 293 mm.Millipore filter (membranes used: 1.2 microns, 0.45 micron, and 0.3micron).

The liquid blood plasma product prepared in the above manner by thesuccessive fractionation, first by cryoprecipitation and then by glycineprecipitation, has an AHF concentration of high potency. This liquidproduct is then frozen by shell freezing (=60 C.) and storing in a flashfreezer C. to 30 C.) for at least three hours. The frozen product canthen be retained under ordinary refrigeration conditions (54 C.,preferably at 20 C. to '30 C.) without loss of its AHF activity forperiods of time of one year and longer. This product when reconstitutedfor administration contains five times the AHF activity of an equalvolume of normal blood plasma and is contained in only one-fifteenth theamount of protein present in plasma providing an equal amount of AHFactivity. The reconstituted product can be administered intravenously tohemophiliacs as required by conventional transfusion means.

EXAMPLE 2 Example 1 is repeated up to and including the step of addingthe citrated saline to the glycine precipitate. The redissolvedprecipitate is adjusted to pH 6.5 with 1.0 normal acetic acid.Polyethylene glycol 4000 is added to the solution to make the PEGconcentration 3.5 percent. The mixture is gently agitated at roomtemperature for ten minutes, and then centrifuged for fifteen minutes at5000 r.p.m. The supernate is decanted and adjusted to pH 6.88 with 1.0normal sodium hydroxide. Additional polyethylene glycol 4000 is added tothe solution to make the final PEG concentration 10 percent. The mixtureis gently agitated at room temperature for thirty minutes, andcentrifuged at 5000 r.p.m. for one-half hour. The supernate is decantedand the precipitate is washed in cold water (2 C.). Spin washing is thencarried out for five minutes at 5000 r.p.m. at a temperature of 4 C. Thesupernate is decanted and the precipitate is dissolved in citratedsaline. The clarified solution is filtered using a 293 mm. Milliporefilter as described in Example 1. This final liquid product can befrozen by shell freezing, followed by storage in a flash freezer for atleast three hours, and then retained under ordinary refrigerationconditions for subsequent use in hemophiliac therapy in the manner ofthe final product of Example 1.

EXAMPLE 3 Example 2 is repeated including the additional step ofpurification of the AHP fraction with ECTEOLA cellulose resin in thefollowing manner.

Reagents ECTEOLA cellulos resin (1) Mix 60 gm. NaOH with 150 ml. H 0.

(2) Allow mixture to cool.

(3) Place 60 gm. cellulose (Whatman Cellulose Powder CF 11) in a beakerand mix thoroughly with above NaOH solution.

(4) Allow mixture to stand overnight (12 hours).

(5) Next day prepare a solution of 35 ml. triethanolamine and 60 ml. ofepichlorohydrin. Mix well under a hood.

(6) Add this solution quickly to the above cellulose and miX Well. Placethe reaction vessel out of draft. (The reaction is exothermic and willheat to about 100 C. The mixture will turn brown in one to two hours.)

(7) Cool the mixture at room temperature under hood.

(8) Add 350 ml. 2 M NaCl in small portions.

(9) Filter this mixture through a coarse sintered glass filter.

(10) Wash precipitate twice with 500 ml. of l N NaOH.

(This removes deep discoloration.)

(ll) Suspend precipitate in 350 ml. 1 N HCl in filter funnel. Applyvacuum.

(12) Repeat step 11 with 250 ml. 1 N NaOH.

(13) Repeat step 11 un'th 250 ml. N HCl.

(14) Repeat step 11 with 250 ml. 1 N NaOH.

(15) Transfer precipitate to a 3 liter beaker.

(16) Add 250 ml. 1 N NaOH. Mix.

(17) Add distilled water to mixture to fill beaker; mix.

Cover and allow to stand overnight.

(18) Decant supernatant.

(19) Add water to precipitate to fill beaker and mix.

(20) Wash mixture on the filter with water. (Four liters or more, untila negative test for alkali with 1% alcoholic phenolphthalein isobtained.)

(21) Make a final wash with two 250 ml. portions absolute ethanol.

(22) Place on filter paper. Mash and spread and place it to dryovernight.

Chloride buffer 0.8% NaCl (8 gm./l.) 0.02 M Imidazole (1.36 gm./l.)Adjust pH to 6.9 with l N HCl Elutting butfer 0.5 M NaCl 0.02 MImidazole (1.36 gm./l.) Adjust pH to 6.9 with 1 N HCl A commerciallyavailable ECTEOLA cellulose resin which is first recycled with NaOH, forexample, as in the following manner, can be used in place of theabove-prepared ECTEOLA cellulose resin.

Recycled commercial ECTEOLA cellulose resin- (l) Mix gm. commercialECTEOLA cellulose resin with 350 ml. 2 M NaCl.

(2) Filter this mixture through a coarse sintered glass filter.

(3) Wash precipitate two times with 500 ml. 1 N NaOH.

(4) Wash one time with 350 ml. 1 N HCl.

(5) Wash one time with 250 ml. 1 N NaOH.

(6) Wash one time with 250 ml. 1 N HCl.

(7) Wash one time with 250 ml. 1' N NaOH.

(8) Transfer precipitate to a 3 liter beaker and add 250 ml. 1 N NaOH.Mix.

(9) Add distilled water to mixture to fill beaker. Mix.

Cover and let stand overnight.

(10) Decant liquid; add 3 liters water to precipitate; mix.

Filter.

(11) Wash precipitate with water until phenolphthalein test is negative.

(12) Wash precipitate two times with 500 ml. absolute EtOH.

(l3) Air-dry precipitate on filter paper.

7 Procedure When the starting material for the purification with ECTEOLAcellulose resin is the cryoprecipitate concentrate of AHF or thepolyethylene glycol-precipitated fraction of AHF, the AHF fraction isfirst dissolved in chloride buffer. When the starting material is theglycineprecepitated fraction of AHF, the AHF fraction is first dialyzedagainst the chloride buffer for one hour to remove glycine and reduceits ionic strength. Purification of the buffered AHF fraction by columntechnique with the above-prepared ECTEOLA cellulose resin proceeds asfollows:

The ECTEOLA cellulose resin is equilibrated overnight (12 hours) in a C.box by mixture with chloride buffer in proportions of 15 gm. resin to600 ml. buffer. The resulting resin slurry is poured on a column of fromone to 1 /2 inches in diameter x 18 inches high. After the buffer comesdown to the level of the resin, the AHF fraction is adjusted to 2 /2 ml.per minute and the amount of AHF fraction applied to a single column isfrom 1000 to 2500 units of AHF. (One unit of AHF is equal to the AHFactivity in one cc. of normal human blood plasma.) After the AHF hasbeen applied to the column, from 200 to 500 ml. chloride buffer iswashed through the resin. When the chloride buffer comes down to thelevel of the resin, the eluting buffer is applied to the column. Theeluate is collected in ten ml. portions and analyzed for proteinfibrinogen and AHF activity.

The eluate portions having the most active AHF activity are retained andstabilized by the addit on of 1% albumin. The stabilized solution isthen filtered using a 293 mm. Millipore filter as in Example 1. A silverfilter of the same size can also be used in place of the Milliporefilter. This final liquid product can then be frozen by shell freezing,followed by storage in a flash freezer for at least three hours, andthen retained under ordinary refrigeration conditions in the manner ofthe final product of Example 1.

EXAMPLE 4 Example 1 is repeated up to and including the step ofdissolving the cryoprecipitate in glycine citrated saline. The dissolvedcryoprecipitate is adjusted to pH 6.5 with 0.1 normal acetic acid.Polyethylene glycol 4000 is added to the solution to make the PEGconcentration 3.5 percent. The mixture is gently agitated at roomtemperature for ten minutes, and then centrifuged for fifteen minutes at5000 r.p.m. The supernate is decanted and adjusted to pH 6.88 with 0.1normal sodium hydroxide. Additional polyethylene glycol 4000 is added tothe solution to make the final PEG concentration percent. The mixture isgently agitated at room temperature for thirty minutes, and centrifugedat 5000 r.p.m. for one-half hour. The supernate is decanted and theprecipitate is Washed in cold water (2 0). Spin washing is then carriedout f r five minutes at 5000 r.p.m. at a temperature of 4 C. Thesupernate is decanted and the precipitate is redissolved in citratedsaline.

The redissolved precipitate is adjusted to pH 6.88 with 0.1 normalacetic acid and then reprecipitated with glycine according to theprocedure of Example 1 except that the molarity of the glycine reagentis 1.8. The glycine precipitate is washed and clarified, and then frozenas in Example 1.

The AHF concentrate prepared by the procedure of this example containsless than 0.05% (generally as little as 0.01%) residual polyethyleneglycol Whereas the AHF concentrate prepared by the procedure of Example2, above, contains on the order of about 0.5% polyethylene glycol. Thefinal product made by the procedure in this example also issubstantially more soluble than the like product prepared in Example 2,above. This concentrate has more than thirty times the AHF activity ofan equal volume of plasma; and the AHF activity of this concen trate iscontained in less than one hundredth the amount of protein present inplasma providing an equal amount of AHF activity.

Various modifications and adaptations of the present invention can bedevised, after reading the foregoing specification and the claimsappended hereto, by the person skilled in the art without departing fromthe spirit and scope of the invention. All such variations andmodifications are included within the scope of the invention as definedin the following claims.

What is claimed is:

1. In the method of preparing a concentrate of AHF, the improvementcomprising the fractionation of a cryoprecipitate concentrate of AHFwith both polyethylene glycol and glycine, said cryoprecipitateconcentrate of AHF being obtained as the precipitate from the freezingof blood plasma at 24 C. followed by thawing and separated from thesupernatant, said fractionation with polyethylene glycol comprising twosuccessive precipitations with polyethylene glycol having a molecularweight of from about 200 to about 20,000, first to a concentration offrom about 3% to about 4% by weight of polyethylene glycol followed bydiscardal of the resulting precipitate and recovery of the supernate,and then to a concentration of about 10% by weight of polyethyleneglycol followed by recovery of the resulting precipitate and discardalof the supernate, said fractionation with glycine comprisingprecipitation with aqueous glycine having a molarity of from about 1.3to about 1.8 followed by recovery of the resulting precipitate anddiscardal of the supernate.

2. The method of Claim 1 in which the fractionation with polyethyleneglycol precedes the fractionation with glycine.

3. The method of claim 2 in which the glycine has a molarity of about1.8.

4. The method of claim 1 in which the polyethylene glycol has an averagemolecular weight of from about 400 to about 6000.

5. The method of claim 1 in which the polyethylene glycol has an averagemolecular weight of about 4000.

6. The method of claim 1 including the additional step of purificationwith triethanolaminoethylated cellulose resln.

7. The method of claim 1 in which the fractionation with polyethyleneglycol precedes the fractionation with glycine, the polyethylene glycolhas an average molecular weight of about 4000 and the glycine has amolarity of about 1.8.

8. The method of claim 7 including the additional step of purificationwith triethanolaminoethylated cellulose resin.

References Cited UNITED STATES PATENTS 2,867,567 1/1959 Bidwell 4241773,415,804 12/1968 Polson 260l 12 FOREIGN PATENTS 883,549 11/1961 GreatBritain.

OTHER REFERENCES Chem. Abstracts, vol. 58, 1963, 4380d, Jorpes et al.

Chem Abstracts, vol. 59, 1963, 6784g, Michael et al.

Chem. Abstracts, vol. 61, 1964, 9705h-9706a, Wagner et al.

Nature, vol. 203, 1964, p. 312, Pool et al.

American I. of the Medical Sciences, vol. 250, 1965, pp. 643-50, Websteret al.

Chem. Abstracts, vol. 64, February 1966, 7133b, Wagner et al.

Journal of the American Medical Association, vol. 199, February 1967,pp. 554-8, Simson et al.

Journal of Hematology (Blood), vol. 28, p. 1011, Dec. 5, 1966, Johnsonet al.

HOWARD E. SCHAIN, Primary Examiner US. Cl. X.-R. 424--177

